Spindle component 25 predicts the prognosis and the immunotherapy response of cancers: a pan-cancer analysis

Spindle component 25 (SPC25) is one of the four proteins that make up the nuclear division cycle 80 (NDC80) complex, the other three components being Ndc80p, Nuf2p, and spindle component 24. Deregulation of the components of this complex can lead to uncontrolled proliferation and reduced apoptosis. However, the prognostic and immunotherapeutic value of SPC25 in pan-cancer remains unclear. Data from the UCSC Xena, TIMER2.0, and TCGA were analyzed to investigate the overall differential expression of SPC25 across multiple cancer types. The survival prognosis, clinical features, and genetic changes of SPC25 were also evaluated. Finally, the relationship between SPC25 and immunotherapy response was further explored through Gene Set Enrichment Analysis, tumor microenvironment, and immune cell infiltration. The transcription and protein expression of SPC25 were significantly increased in most cancer types and had prognostic value for the survival of certain cancer patients such as ACC, CESC, KIRC, KIRP, LIHC, LUAD, MESO, STAD, THYM, and UCEC. In some cancer types, SPC25 expression was also markedly correlated with the TMB, MSI, and clinical characteristics. Gene Set Enrichment Analysis showed that SPC25 was significantly associated with immune-related pathways. In addition, it was also confirmed that the expression level of SPC25 was strongly correlated with immune cell infiltration, immune checkpoint genes, immune regulatory genes, Ferroptosis-related genes, Cuproptosis-related genes, and lactate metabolism-related genes. This study comprehensively explored the potential value of SPC25 as a prognostic and immunotherapeutic marker for pan-cancer, providing new direction and evidence for cancer therapy.

Undoubtedly, the global burden of cancer poses a monumental challenge to public health, obstructing the pursuit of enhanced longevity 1,2 .The realm of cancer treatment continues to grapple with grave circumstances.Tumors, notorious for their intricate biology, exhibit an array of processes-proliferation, evasion of growth inhibitors, resilience to cell demise, angiogenesis stimulation, invasion activation, and metastasis 3 .In addition, the dynamic interplay between tumor invasion and the host's immune response bears a striking correlation to the progression of cancer.As a critical player in tumor control, the immune system can facilitate efficacious immunotherapy by leveraging preexisting adaptive immune responses within the tumor, such as those involving checkpoint inhibitors 4 .In light of the widespread nature of tumors and the convoluted oncogenesis process, delving into the expression levels of pan-cancer-associated genes holds immense potential for advancements in clinical treatment and prognostic prediction.
The nuclear division cycle 80 (Ndc80) complex, vital to chromosome segregation, comprises four components: Spindle component 25 (SPC25), Ndc80p, Nuf2p, and Spindle component 24 (SPC24) 5 .Of these, SPC25 plays a pivotal role in orchestrating mitotic processes, forming heterodimers with SPC24 to regulate microtubule-centromeric attachment, chromosome organization, and spindle checkpoint activation 5,6 .Studies have revealed that aberrant chromosome segregation and compromised checkpoint signaling culminate in chromosomal instability (CIN)-a characteristic hallmark of numerous cancer types 7 .This phenomenon frequently arises during the initial stages of tumorigenesis, leading researchers to postulate that CIN may constitute the primary catalyst for tumor initiation 8,9 .Recent investigations have unveiled a noteworthy association between SPC25 dysregulation

Tumor microenvironment and immune infiltrate analysis
To analyze the infiltration levels of immune cells and stromal cells in pan-cancer, we used ESTIMATE to determine the correlation between SPC25 expression and immune score, stromal score.Moreover, the relationship between SPC25 expression level and different immune cells (CD8 T cells and monocytes, etc.) was analyzed using the CIBERSORT method (http:// ciber sort.stanf ord.edu/) and TIMER2.0database (http:// timer.cistr ome.org/).The p filter was set to less than 0.001 [26][27][28] .

Co-expression analysis of the SPC25 gene
At the same time, the correlation between SPC25 and other genes was evaluated.These genes contained Cuproptosis-related genes, Ferroptosis-related genes, lactate metabolism-related genes, and immune chemokines genes.Heatmaps were used to show the results of the co-expression analyses.
Co-expression gene set source: (1) lactate metabolism related genes

The relationship between SPC25 and clinical information
In the advanced stages of ACC and KIRP, especially in stage III and stage IV, the expression of SPC25 was significantly higher than early stages.In KICH, the expression of SPC25 was the highest in stage IV, which was markedly different from stages I, II, and III.It might be linked to excessive proliferation, poor prognosis, invasion of stage IV cancer cells, and cell death inhibition.In stage II, stage III, and stage IV of LUAD and KIRC, SPC25 was higher compared to stage I.However, the sample size of stage IV LUAD was too small to effectively indicate the comparative results.Furthermore, the expression of SPC25 was higher in stages I of THCA and SKCM.SPC25 expression in ESCA, KIRP, and LAML was significantly higher in patients under 65 years of age than in patients over 65 years of age.In contrast, SPC25 expression in STAD, LUSC, and THYM was significantly higher in patients older than 65 years than in patients younger than 65 years (Fig. 4, Supplementary Fig. 5).

Relationship of SPC25 with TMB and MSI
More and more studies show that TMB and MSI may be independent biomarkers reflecting the therapeutic effect of immune checkpoint inhibitors and the prognosis of cancers 29,30 .Thence, we further explored the relationship of SPC25 with TMB and MSI in the pan-cancer cohort.SPC25 expression was positively related to the TMB in ACC, UCS, UCEC, STAD, SKCM, SARC, PRAD, OV, MESO, LUSC, LUAD, LGG, KIRC, KICH, HNSC, BRCA, and BLCA, whereas the negative association was observed in THYM.For MSI, a positive association was obtained in UCEC, STAD, SARC, READ, ESCA, and HNSC, the negative association was shown in DLBC, THYM, LAML, and KICH (Fig. 5).

GSEA analysis of SPC25
Single-gene GSEA was used to identify relevant pathways affected by SPC25 expression in pan-cancer.The results showed that SPC25 is positively correlated with the immune-related pathways in ESCA, THCA, PCPG, and GBM.SPC25 is positively correlated with HNSC, KIRC, LGG, LIHC, and OV signal pathways, such as cell division, spindle filament activity, and smooth muscle cell activity.Also, SPC25 was positively correlated with the chromosome activity, nuclear organization, and cell metastasis of PRAD, STAD, and THYM (Supplementary Fig. 1).Furthermore, we observed that SPC25 is negatively correlated with cell metabolism, NK cell activity, and vascular regulation in DLBC, GBM, ESCA, SARC, and PCPG, and negatively correlated with READ and ECEC signal pathways, such as axoneme activity, vascular-associated smooth muscle activity, and zymogen activation.Our findings suggested that SPC25 generally correlated with many important pathways in cancer formation (Fig. 6).

Correlation between SPC25 expression and immune infiltrating level in pan-cancers
According to GSEA, we observed a potential association between SPC25 and immune-related factors.Therefore, we further performed tumor microenvironment and immune infiltrate analysis.The results showed that SPC25 had a positive correlation with the immune score in KIRC (R = 0.22), THYM (R = 0.29), and THCA (R = 0.4), while SPC25 was a negative correlation with the immune score in ACC (R = − 0.   score: R = − 0.16), SPC25 transcript levels were consistently negatively correlated with immune and stromal scores (Fig. 8).Immune-related cell infiltration is the main mechanism affecting the tumor microenvironment.Therefore, we further studied the relationship between SPC25 expression and immune infiltration analysis in pan-cancer.We found that SPC25 was positively correlated with macrophages in ACC, LAML, LGG, and LUAD, while was negatively correlated with KIRP, and LIHC.On the other hand, SPC25 showed a correlation with T cells in BLCA, BRCA, COAD, DLBC, KIRC, and KIRP.In addition, SPC25 is negatively correlated with mast cells in BRCA and neutrophils in HNSC (Fig. 9).

SPC25 correlated with the majority of Cuproptosis-related genes, Ferroptosis-related genes, lactate metabolism-related genes, and immune chemokines genes
In conclusion, SPC25 may have a significant relationship with tumor immune regulation, so we further explored the role of SPC25 at the gene level.We performed comprehensive co-expression analysis on the Cuproptosisrelated genes, Ferroptosis-related genes, lactate metabolism-related genes, and immune chemokines genes.The results showed that immune chemokines genes 47 immune checkpoint genes showed that most genes in LIHC, THCA, and THYM were significantly positively correlated with SPC25, while negatively correlated in GBM, LGG, PRAD, STAD, and UCEC.The results showed that Ferroptosis-related genes such as SLC3A2, HSF1, and SLC7A11 were significantly positively correlated with SPC25 in pan-cancer.In Cuproptosis-related genes, SPC25 was positively correlated with GCSH, CDKN2A, PDHB, PDHA1, DLAT, DLD, and SLC31A1 of pan-cancer.TWNK, TRMU, RNASEH1, PIF1, PDSS1, MGME1, DNA2, and DARS2 in lactate metabolism-related genes were significantly correlated with the expression of SPC25 in pan-cancer.Similarly, we can see that most of the lactate metabolism-related genes in BLCA, BRCA, ESCA, HNSC, LGG, LIHC, SKCM, and UVM are closely related to the expression of SPC25 (Supplementary Fig. 6).

Discussion
Through an exhaustive examination, our research illuminated the prognostic and potential immunotherapeutic significance of SPC25 in the context of pan-carcinoma.We observed markedly elevated gene levels of SPC25 in the majority of tumors, with some cancers demonstrating distinct prognostic implications.Moreover, the expression of SPC25 bears a strong connection to immune and inflammatory pathways, immune cell infiltration, and an array of immune-associated genes.Consequently, SPC25 emerges as a potential prognostic biomarker and a predictor of immunotherapy outcomes.SPC25, an integral component of the NDC80 complex, plays a pivotal role in assembling the microtubulebinding domain of kinetochores and facilitating chromosome alignment with the metaphase plate.In concert with SPC24, SPC25 orchestrates chromosome alignment and spindle formation, processes indispensable for chromosome segregation 31 .As a crucial element, it auto-inhibits the interaction between the microtubule and the Ndc80 complex, thereby regulating chromosome segregation during cell mitosis-a critical process intertwined with tumorigenesis 32,33 .Hitherto, research on SPC25 has primarily centered on its role in cell mitosis, where dysregulation has been implicated in abnormal cell division.Data concerning the relationship between SPC25 and cancer development remain scarce.Nonetheless, statistical analyses drawing upon online databases, such as TCGA, insinuate a close association between SPC25 expression and poor prognosis in patients with hepatocellular carcinoma (HCC), non-small cell lung adenocarcinoma cells, and prostate cancer.Preliminary www.nature.com/scientificreports/ in vitro and in vivo functional tests of tumor cells have corroborated that elevated SPC25 expression can stimulate HCC cell proliferation and augment cancer stem cell (CSC) characteristics 14,34 .In light of this evidence, our focus gravitates toward the mechanisms underpinning SPC25's involvement in multiple tumors, delving deeper at the pan-cancer level.Upon evaluating SPC25 mRNA levels in 33 human tumors across multiple databases, we discerned a significant reduction in expression across the majority of tumors.Contrasting transcription levels, we found that BLCA, BRCA, CESC, CHOL, COAD, ESCA, GBM, HNSC, KIRC, KIRP, LIHC, LUAD, LUSC, PRAD, READ, STAD, THCA, and UCEC exhibited markedly elevated transcription levels.For most cancers, SPC25 expression exhibited no significant discrepancies based on clinical stage, age, or sex.However, in certain cancers, particularly ACC, KICH, KIRC, KIRP, LIHC, MESO, UCEC, and UVM, SPC25 expression emerged as an independent prognostic factor and a potential prognostic marker.
Furthermore, GSEA enrichment findings revealed that SPC25 exhibited a strong association with immune and inflammatory responses, protein metabolism, spindle filament activity, and chromosome activity, aligning with established tumor mechanisms.We honed in on various immune pathways, encompassing inflammatory responses, T-cell reactions, and macrophage response signaling pathways, which are intimately linked to tumorigenesis.To delve deeper into SPC25's potential value, we probed its relationship with the tumor microenvironment and immune cell infiltration.Our results uncovered a robust correlation between SPC25 and the infiltration of diverse immune cell types (such as CD4 + T cells, macrophages, and T cell regulation) within multiple tumor TMEs.Consequently, we surmise that SPC25 may modulate immune cells in the tumor microenvironment via numerous pathways, as opposed to exclusively targeting specific immune cells.
Ordinarily, the immune system employs immune cell infiltration to identify and eradicate tumor cells within the TME.The infiltration of immune cells and the elicitation of anti-tumor immune responses are chiefly governed by an array of chemokines, chemokine receptors, cytokines, and immune checkpoints.Bearing these factors in mind, we assessed the connection between SPC25 and immune checkpoint genes.Intriguingly, SPC25 exhibited a significant association with the majority of genes in BLCA, KIRC, LIHC, THCA, and THYM malignancies, aligning closely with prior GSEA enrichment outcomes.This intimates that SPC25 could function as a potential regulatory target in the immunotherapy of these cancers and suggests its involvement in inducing immune cell recruitment is far from trivial [35][36][37] .The tumor immune microenvironment plays an important role in tumor development, prognosis, and immunotherapy 38,39 .In ACC and COAD, SPC25 was significantly negatively correlated with immune score and immune cell infiltration.However, different immune cell types also play different roles in cancer, for example, CD8 + T cells tend to be associated with a good prognosis, while regulatory T cells are mostly associated with a poor prognosis 4 .Immune cell infiltration analysis showed a significant negative correlation between SPC25 and regulatory T-cell infiltration, suggesting that COAD patients with high SPC25 expression may have a better prognosis, which is also consistent with our analysis results.COAD is a rare but highly malignant tumor, and treatment options for advanced cancers are severely limited.Therefore, immunotherapies such as checkpoint inhibitors and monoclonal antibodies may eventually be accepted as effective potential treatments for these patients 40 .Importantly, SPC25 showed a significant positive correlation with COAD expressing many immune checkpoint genes, especially NRP1, CD276, TNFSF14, and other important immunotherapeutic targets, which may also be closely related to the immune escape mechanism of ACC tumor cells.

Conclusion
We found that SPC25 is a novel biomarker for a variety of cancers.We found that SPC25 was significantly associated with prognosis, mutation, and immunity in pan-cancer.It promises to be a new therapeutic target for many types of cancer.

Data availability
The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Figure 1 .
Figure 1.Differences in SPC25 expression between normal and tumor tissue samples (A) expression of SPC25 in pan-cancer and corresponding normal samples in TIMER2.0 (B) normal colon tissue and colon cancer (C) SPC25 cell location, green indicates the distribution of SPC25.The red box represents the tumor (T) and the blue box represents normal tissue (N).

Figure 2 .
Figure 2. Prognostic assessment of SPC25 expression in OS, DFI, DSS and PFI.Correlation between SPC25 expression and OS (A) DFI (B) DSS (C) PFI (D) by utilizing Cox.HR (hazard ratio) > 1 indicates that SPC25 may be an adverse factor in the occurrence and development of cancer; 0 < HR < 1 indicates that SPC25 may be a protective factor in cancer.

Figure 3 .Figure 4 .
Figure 3. Kaplan-Meier analysis of OS in patients with high and low SPC25 expression (A-G) high expression of SPC25 predicted poor OS than low expression (H-J) high expression of SPC25 predicted better OS than low expression.

Figure 9 .
Figure 9.The correlation between SPC25 expression and immune cell infiltration (A-T) Scatter plots showing that tumor-infiltrating immune cells were significantly correlated with SPC25 expression. )